Gold standard assay techniques: Pros and cons

ELISA

Enzyme-linked immunosorbent assay (ELISA) is a singleplex assay technique carried out in 96- or 384-well plates. It plays a major role in the qualitative and quantitative identification of analytes. Direct and indirect ELISA detection methods are available, each of which have their own benefits and drawbacks. For instance, specific analytes present in a crude solution can be accurately measured using direct ELISA. ELISA detection is typically rapid, owing to it requiring just one single antibody and only involving a few steps. Indirect ELISA detection is more versatile; primary antibodies can often be generated in one species and subsequent detection can be carried out using the identical labeled secondary antibody.

Compared with multiplexing, ELISA takes longer to measure multiple analytes, since many workflows occur concurrently. Indirect ELISA detection requires an additional incubation step, which further reduces efficiency in terms of time spent in the laboratory.

Western blotting

In western blotting, a tissue sample is examined and specificity is calculated via band positioning. This sensitive immunological protein detection strategy has the potential to detect proteins as small as 0.1ng, with only the target protein being detected. This selectivity may help researchers to analyze only the protein of interest in a complex solution. The specificity of this technique is also important – a sample will be sorted into the different proteins present in the solution using gel electrophoresis, thereby offering straightforward detection.

The western blot method is specific, but it may produce a false-negative result if adequate time is not allowed for larger proteins to transfer through the membrane. Faded or multiple bands caused by the blotting itself may give unfavorable and hard-to-interpret results. Moreover, it is difficult to automate the western blot method and the transfer of larger proteins can be challenging.

Radioimmunoassay

Radioimmunoassay is an in vitro process involve the use of a radioisotope-labeled antigen to measure antibody radioactivity. Gamma-radioactive iodine isotopes are typically used to label the antigens. The radioimmunoassay method, like western blotting, is highly specific and sensitive. It is also not limited to only serum antigens. Theoretically, it is possible to use any biological molecule in a radioimmunoassay.

The use of radiolabeled reagents is associated with potential radiation hazards, often meaning that only specially trained individuals can carry out this assay. Laboratories generally require licensure to use this radioactive material, as well as having to follow special protocols for its storage and disposal.

Multiplex assays: Pros and cons

Newer technological advancements have resulted in the development of more robust multiplex assays in the detection of complex diseases. Dozens of proteins can be detected, identified, and monitored simultaneously using multiplex assays, thereby optimizing clinical research and paving the way for the discovery of new therapeutic options.

Bead- and planar-based assays are the two basic types of multiplex assays. Fluorescent bead microarray, DNA microarray, antibody microarray, and protein microarray are examples of the specific types of multiplex assay techniques available.

Pros

Compared to singleplex assays such as the western blot or ELISA, multiplex assays enable researchers to acquire more data from their samples. The ability to evaluate multiple analytes at the same time means procedure times are reduced, providing savings on time and cost. Multiplexing can also be automated and customized, further reducing procedure times. Furthermore, time and cost efficiency is also enhanced because a combination of assays can be carried out for multiple analytes of different types, all in a single reaction volume.

Cons

The need for a validated antibody pair and specialized equipment are the disadvantages to using a multiplex assay technology. A multiplex system may also require higher initial investment compared with singleplex devices.

Ther-Mix for immunoassays

Incubation on a shaker is usually needed for immunoassay techniques. The techniques also involve mixing and heating for varying amounts of time and at different temperatures. The use of mixers, shakers, stopwatches, incubators and various other tools can make procedures cumbersome and time-consuming.

Vitl’s Ther-Mix is a heated mixer that enables users to program and save their custom mixing programs. Once the required program is set on the Ther-Mix, the mixer can automatically run through the different mixing and incubation steps, without it needing constant attention. The Ther-Mix alerts the operator on completion of the protocol. The mixer has interchangeable Heated Modules that allow operators to use different types of sample plates and tubes.

To learn more about our Ther-Mix Microplate and Tube Mixer, visit its product page.

References

• Tighe PJ, Ryder RR, Todd I, Fairclough LC. ELISA in the multiplex era: potentials and pitfalls. Proteomics Clin Appl. 2015;9(3-4):406-422.

• Immunoassays: Protein Arrays vs. ELISA and Westerns. Grace Bio-Labs. https://gracebio.com/immunoassays-protein-arrays-vs-elisa-and-westerns/.

• Five Ways That Multiplex Technology Accelerates Cancer Research. Luminex White Paper. http://cdn2.hubspot.net/hub/128032/file-1242255217-pdf/WP885.xMAP.Cancer.0714.WR.pdf?t=1410627766815.

• The Role of Multiplexed Assays in Vaccine Development. Luminex White Paper. http://www.nature.com/app_notes/nmeth/2015/151011/pdf/an9676.pdf?spMailingID=50203593&spUserID=ODkwMTM2NjMzOQS2&spJobID=821232547&spReportId=ODIxMjMyNTQ3S0&foxtrotcallback=true.

• The power of multiplexing. ThermoFisher Scientific White Paper. https://www.thermofisher.com/content/dam/LifeTech/global/technical-reference-library/s2s/dbourdon/The%20power%20of%20multiplexing.pdf.

• Multiplex immunoassay techniques: a review of current methods. Abcam. http://www.abcam.com/kits/multiplex-immunoassay-techniques-review-of-methods-western-blot-elisa-microarray-and-luminex.

• Multiplexed Protein Assays. Biocompare. http://www.biocompare.com/Editorial-Articles/41806-Multiplexed-Protein-Assays/.

• Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 2006;38(4):317-323.